Genetic transformation, in which cells internalize exogenous DNA and integrate it into their chromosome, is widespread in the bacterial kingdom. It involves a specialized membrane-associated machinery for binding double-stranded (ds) DNA and uptake of single-stranded (ss) fragments. In the human pathogen Streptococcus pneumoniae, this machinery is specifically assembled at competence. The EndA nuclease, a constitutively expressed virulence factor, is recruited during competence to play the key role of converting dsDNA into ssDNA for uptake. Here we use fluorescence microscopy to show that EndA is uniformly distributed in the membrane of noncompetent cells and relocalizes at midcell during competence. This recruitment requires the dsDNA receptor ComEA. We also show that under ‘static’ binding conditions, i.e., in cells impaired for uptake, EndA and ComEA colocalize at midcell, together with fluorescent end-labelled dsDNA (Cy3-dsDNA). We conclude that midcell clustering of EndA reflects its recruitment to the DNA uptake machinery rather than its sequestration away from this machinery to protect transforming DNA from extensive degradation. In contrast, a fraction of ComEA molecules were located at cell poles post-competence, suggesting the pole as the site of degradation of the dsDNA receptor. In uptake-proficient cells, we used Cy3-dsDNA molecules enabling expression of a GFP fusion upon chromosomal integration to identify transformed cells as GFP producers 60–70 min after initial contact between DNA and competent cells. Recording of images since initial cell-DNA contact allowed us to look back to the uptake period for these transformed cells. Cy3-DNA foci were thus detected at the cell surface 10–11 min post-initial contact, all exclusively found at midcell, strongly suggesting that active uptake of transforming DNA takes place at this position in pneumococci. We discuss how midcell uptake could influence homology search, and the likelihood that midcell uptake is characteristic of cocci and/or the growth phase-dependency of competence.

Comprehensive analysis of the intranuclear territories and motion of budding yeast chromosome XII loci suggests that long-range chromosome architecture is mainly determined by the physical principles of polymers.

How spatial organization of the genome depends on nuclear shape is unknown, mostly because accurate nuclear size and shape measurement is technically challenging. In large cell populations of the yeast Saccharomyces cerevisiae, we assessed the geometry (size and shape) of nuclei in three dimensions with a resolution of 30 nm. We improved an automated fluorescence localization method by implementing a post-acquisition correction of the spherical microscopic aberration along the z-axis, to detect the three dimensional (3D) positions of nuclear pore complexes (NPCs) in the nuclear envelope. Here, we used a method called NucQuant to accurately estimate the geometry of nuclei in 3D throughout the cell cycle. To increase the robustness of the statistics, we aggregated thousands of detected NPCs from a cell population in a single representation using the nucleolus or the spindle pole body (SPB) as references to align nuclei along the same axis. We could detect asymmetric changes of the nucleus associated with modification of nucleolar size. Stereotypical modification of the nucleus toward the nucleolus further confirmed the asymmetric properties of the nuclear envelope.

Quantification of cardiomyocyte contraction is usually obtained by measuring globally cell shortening from the displacement of cell extremities. We developed a correlationbased optical flow method, which correlates the whole-cell temporal pattern with a precise quantification of the intracellular strain wave at the sarcomeres level. A two-dimensional image correlation analysis of cardiomyocytes phase-contrast images was developed to extract local cell deformations from videomicroscopy time-lapse sequences. Test images, synthesized from known intensity displacement fields, were first used to validate the method. Intracellular strain fields were then computed from videomicroscopy time-lapse sequences of single adult and neonatal cardiomyocytes. The propagation of the sarcomeres contraction-relaxation wave during cell contraction has been successfully quantified. The time-varying patterns of intracellular displacement were obtained accurately, even when cardiomyocyte bending occurred in pace with contraction. Interestingly, the characterization of the successive 2D displacement fields show a direct quantification of the variation with time of intracellular strains anywhere in the cell. The proposed method enables a quantitative analysis of cardiomyocyte contraction without requiring wave tracking with the use of fluorescent calcium probes. Thus, our algorithmic approach provides a fast and efficient tool for analyzing the correlation between global cell dynamical behavior and mechanosensitive intracellular processes.

A precise characterization of cell elastic properties is crucial for understanding the mechanisms by which cells sense mechanical stimuli and how these factors alter cellular functions. Optical and magnetic tweezers are micromanipulation techniques which are widely used for quantifying the stiffness of adherent cells from their response to an external force applied on a bead partially embedded within the cell cortex. However, the relationships between imposed external force and resulting bead translation or rotation obtained from these experimental techniques only characterize the apparent cell stiffness. Indeed, the value of the estimated apparent cell stiffness integrates the effect of different geometrical parameters, the most important being the bead embedding angle 2gamma, bead radius R, and cell height h. In this paper, a three-dimensional finite element analysis was used to compute the cell mechanical response to applied force in tweezer experiments and to explicit the correcting functions which have to be used in order to infer the intrinsic cell Young's modulus from the apparent elasticity modulus. Our analysis, performed for an extensive set of values of gamma, h, and R, shows that the most relevant parameters for computing the correcting functions are the embedding half angle gamma and the ratio h(u)/2R, where h(u) is the under bead cell thickness. This paper provides original analytical expressions of these correcting functions as well as the critical values of the cell thickness below which corrections of the apparent modulus are necessary to get an accurate value of cell Young's modulus. Moreover, considering these results and taking benefit of previous results obtained on the estimation of cell Young's modulus of adherent cells probed by magnetic twisting cytometry (MTC) (Ohayon, J., and Tracqui, P., 2005, Ann. Biomed. Eng., 33, pp. 131-141), we were able to clarify and to solve the still unexplained discrepancies reported between estimations of elasticity modulus performed on the same cell type and probed with MTC and optical tweezers (OT). More generally, this study may strengthen the applicability of optical and magnetic tweezers techniques by insuring a more precise estimation of the intrinsic cell Young's modulus (CYM).

Inherently dynamic, chromosomes adopt many different conformations in response to DNA metabolism. Models of chromosome organization in the yeast nucleus obtained from genome-wide chromosome conformation data or biophysical simulations provide important insights into the average behavior but fail to reveal features from dynamic or transient events that are only visible in a fraction of cells at any given moment. We developed a method to determine chromosome conformation from relative positions of three fluorescently tagged DNA in living cells imaged in 3D. Cell type specific chromosome folding properties could be assigned based on positional combinations between three loci on yeast chromosome 3. We determined that the shorter left arm of chromosome 3 is extended in MATα cells, but can be crumpled in MAT a cells. Furthermore, we implemented a new mathematical model that provides for the first time an estimate of the relative physical constraint of three linked loci related to cellular identity. Variations in this estimate allowed us to predict functional consequences from chromatin structural alterations in asf1 and recombination enhancer deletion mutant cells. The computational method is applicable to identify and characterize dynamic chromosome conformations in any cell type.

Bacterial genomes typically consist of a single chromosome and, optionally, one or more plasmids. But whole-genome sequencing reveals about ten per-cent of them to be multipartite, with additional replicons which by size and indispensability are considered secondary chromosomes. This raises the questions of how their replication and partition is managed without compromising genome stability and of how such genomes arose. Vibrio cholerae, with a 1 Mb replicon in addition to its 3 Mb chromosome, is the only species for which maintenance of a multipartite genome has been investigated. In this study we have explored the more complex genome of Burkholderia cenocepacia (strain J2315). It comprises an extra replicon (c2) of 3.21 Mb, comparable in size to the3.87Mb main chromosome (c1), another extra replicon(c3) of 0.87 Mb and a plasmid of 0.09 Mb. The replication origin of c1 is typically chromosomal and those of c2 and c3 are plasmid-like; all are replicated bidirectionally. Fluorescence microscopy of tagged origins indicates that all initiate replication at mid-cell and segregate towards the cell quarter positions sequentially, c1-c2-p1/c3. c2 segregation is as well-phased with the cell cycle as c1, implying that this plasmid-like origin has become subject to regulation not typical of plasmids; in contrast, c3 segregates more randomly through the cycle. Disruption of individual Par systems by deletion of parAB or by addition of parS sites showed each Par system to govern the positioning of its own replicon only. Inactivation of c1, c2 and c3 Par systems not only reduced growth rate, generated anucleate cells and compromised viability but influenced processes beyond replicon partition, notably regulation of replication, chromosome condensation and cell size determination. In particular, the absence of the c1 ParA protein altered replication of all three chromosomes, suggesting that the partition system of the main chromosome is a major participant in the choreography of the cell cycle.

Computational cell models appear as necessary tools for handling the complexity of intracellular cell dynamics, especially calcium dynamics. However, while oscillating intracellular calcium oscillations are well documented and modelled, a simple enough virtual cell taking into account the mechano-chemical coupling between calcium oscillations and cell mechanical properties is still lacking. Considering the spontaneous periodic contraction of isolated cardiac myocytes, we propose here a virtual cardiac cell model in which the cellular contraction is modelled using an hyperelastic description of the cell mechanical behaviour. According to the experimental data, the oscillating cytosolic calcium concentrations trigger the spatio-temporal variation of the anisotropic intracellular stresses. The finite element simulations of the virtual cell deformations are compared to the self-sustained contractions of isolated rat cardiomyocytes recorded by time-lapse video-microscopy.